Method for producing monoclonal antibodies

ABSTRACT

The present invention relates to methods for producing monoclonal antibodies. In particular, the invention relates to high throughput methods for producing and screening monoclonal antibodies more rapidly than conventional methods.

The present invention relates to methods for producing monoclonalantibodies. In particular, the invention relates to high throughputmethods for producing monoclonal antibodies more rapidly thanconventional methods.

The development of monoclonal antibody-producing cell lines by somaticfusions of B lymphocytes with myeloma cells was first described byKohler and Milstein over 25 years ago (Kohler & Milstein, 1975). Sincethen, monoclonal antibodies have played a central role in theexponential growth of our understanding of human physiology,biochemistry and genetics. Monoclonal antibodies are versatile andsensitive tools for detecting and localising specific biologicalmolecules. Monoclonal antibodies can be made against any cell molecule,enabling that molecule to be identified, localised and purified. In somecases, monoclonal antibodies also help identify the function of themolecules to which they bind.

The diagnostic and therapeutic potential of monoclonal antibodies wasalso quickly realised after the hybridoma technique allowed theirdevelopment in the mid 1970s. Monoclonal antibodies have become keycomponents in a vast array of clinical laboratory diagnostic tests. Inaddition, a large number of licensed drugs contain monoclonal antibodiesand vast numbers of drugs in development are monoclonal antibodies. Theclinical use of monoclonal antibodies has been improved by thedevelopment of chimeric and fully humanised monoclonal antibodies whichminimise side-effects in patients.

However, despite the central role of monoclonal antibodies in thesedevelopments in medicine and molecular biology, the process forproducing and screening monoclonal antibodies has changed little sinceit was first developed by Kohler & Milstein in the mid-1970s. Theapproach most often used to produce a monoclonal antibody against aspecific antigen requires a series of immunisations of mice or rats withan antigen over the course of several weeks to enhance the activationand proliferation of mature B cells producing antigen-specificantibodies. Multiple mice are generally immunised and are testedperiodically for the presence of relevant serum immunoglobulin titresprior to somatic fusion. Following fusion, the supernatants ofhybridomas are screened using immunoassays to identify monoclonalantibodies with a high specificity for the antigen.

The time frame required for developing a monoclonal antibody using thisapproach is generally 3 to 9 months.

The development of RIMMS (repetitive, multiple site immunisationstrategy) has enabled somatic fusions to take place just 8-14 days afterthe initiation of immunisation (Kilpatrick et al, 1997). Thesupernatants of the hybridomas produced can then be screened usingstandard immunoassays, allowing a monoclonal antibody against a specificantigen to be isolated much more quickly.

However, even using RIMMS, the production and screening of monoclonalantibodies against large numbers of different antigens requiresconsiderable time and resources. As more and more novel proteins arediscovered, there is a need for faster and more efficient methods forproducing and screening monoclonal antibodies against these proteins, inorder to allow their further characterisation.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a method for producing a monoclonalantibody, said method comprising the steps of:

-   -   a) introducing at least one candidate antigen into an animal;    -   b) recovering antibody-producing cells from said animal and        rendering these cells into a single cell suspension;    -   c) generating an immortalized cell line from said single cell        suspension;    -   d) screening the supernatant of said immortalized cell line        against a protein chip on which the candidate antigen is        displayed; and    -   e) selecting as said monoclonal antibody, an antibody that binds        to said candidate antigen.

The method of the invention has considerable advantages over the methodsof producing monoclonal antibodies that are currently available. Asalready discussed, current methods for producing monoclonal antibodiesagainst more than one antigen involve laborious immunisation andisolation protocols for each individual antigen. In contrast, in themethod of the invention, the animal may be injected with multipleantigens resulting in the simultaneous production of monoclonalantibodies against multiple antigens and increasing the speed andefficiency of monoclonal antibody production. The use of a protein chipin the method of the invention accelerates the process of screening todetect monoclonal antibodies that bind to the antigen or antigens withwhich the animal has been injected. In addition, the protein chip ismore sensitive than conventional screening assays, such as enzyme linkedimmunosorbent assays (ELISAs), resulting in an improved detection ratefor slow secreting hybridoma cells which would be missed usingconventional screening methods. Additionally, the use of a protein chipin the method of the invention enables each supernatant to be screenedmultiple times against an antigen and uses only a fraction of the amountof antigen required for a single screening in a conventional screeningassay such as an ELISA. For example, each supernatant can be screened induplicate, triplicate or quadruplicate against an antigen.

The animal in step a) of the method of the invention may be anynon-human mammalian animal. Preferably, the animal is a mouse, rat,rabbit, hamster or guinea pig. Preferably, the animal is a mouse.

The candidate antigen in step a) is preferably a purified candidateantigen. Either a purified candidate antigen or a mixture of purifiedcandidate antigens may be introduced into the animal. By “purifiedcandidate antigen” is meant that the antigen is a homogenous preparationof antigen that is substantially free from any other components. By “amixture of purified candidate antigens” is meant that more than onepurified antigen is present in the composition used for immunisation,but that the preparation is free from contaminating components for whichthere is no intention to elicit the production of antibodies. Forexample, although using conventional procedures, an animal may beimmunised with multiple antigens simply by immunisation with homogenisedtissue, such immunisation does not represent immunisation with purifiedcandidate antigens as this is defined herein, since the antigens wouldbe contaminated with cellular debris.

Any number of purified candidate antigens may be introduced into theanimal. Preferably, between 1 and 50 purified candidate antigens areintroduced into the animal. Preferably, more than one purified candidateantigens are introduced into the animal. For example, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50 or more than 50 purified candidate antigensmay be introduced into the animal. The antigens may be introducedsimultaneously, in the sense that they are all mixed together.Alternatively, the antigens may be introduced separately one after theother. The introduction of different antigens may be separated by a timeperiod of days. Preferably, the period separating the introduction ofdifferent antigens is less than 48 hours, preferably less than 24 hours.Preferably, the method of introduction involves injection of theantigen(s) into the animal.

By the term “candidate antigen” is meant any substance capable ofinducing an immune response in an animal when that candidate antigen isintroduced into the animal. The term therefore includes proteinaceoussubstances and non-proteinaceous substances.

Proteinaceous substances which are antigens include proteins andderivatives thereof, such as glycoproteins, lipoproteins andnucleoproteins and peptides. Fragments of such proteinaceous substancesare also included within the term “antigen”. Preferably, such fragmentsconsist of or comprise antigenic determinants. Non-proteinaceoussubstances which are antigens include polysaccharides,lipopolysaccharides and nucleic acids. In particular, the term “antigen”includes nucleic acid molecules that induce an immune response againstthe proteins they encode. Fragments of such non-proteinaceous substancesare also included with the term “antigen”. The term “antigen” furtherincludes proteinaceous or non-proteinaceous substances linked to acarrier which are able to induce an immune response, such as lipids orhaptens upon which antigenicity is conferred when they are linked to acarrier. The antigens of the invention may be naturally occurringsubstances or may be synthesised by methods known in the art.

Where one purified antigen is used for immunisation, the antigen ispreferably a proteinaceous substance or a nucleic acid molecule. Wherethe purified antigen is a proteinaceous substance, it may be introducedalone or in the form of a fusion protein. In particular, the inventionprovides that the antigen may be in the form of a fusion proteinexpressed on the surface of a recombinant virion with the animal beinginjected with the recombinant virion. The production of such recombinantvirions using a nucleic acid sequence encoding the proteinaceousantigen, is described in Lindley et al, 2000.

Where more than one purified antigen is used for immunisation, anycombination of purified antigens may be used. The animal may be injectedwith only proteinaceous antigens, only non-proteinaceous antigens, or amixture of both. According to one embodiment of the invention, thepurified antigens are all proteinaceous. The purified antigens may befragments derived from the same protein or different proteins. Thepurified antigens may be recombinant virions derived from a cDNAlibrary, each recombinant virion expressing a protein encoded by a cDNAfrom the library on its surface.

According to a further embodiment, the invention provides that multiplepurified antigens are introduced into the animal in the form of nucleicacid molecules encoding proteins against which it is desired to producemonoclonal antibodies. The nucleic acid molecules may be in DNAmolecules, cDNA molecules or RNA molecules. Preferably, in this aspectof the invention, a cDNA library may be introduced into the animal. Itis therefore possible to inject the animal with nucleic acid moleculesencoding a protein of unknown identity and as described below, cellchips may be used to isolate an antibody against the protein which inturn allows the protein to be purified.

Where the purified antigen is a nucleic acid molecule, it preferablyconsists of or comprises a DNA, cDNA or RNA sequence encoding a proteinagainst which an immune response is to be induced. The nucleic acidmolecule may be a naked nucleic acid molecule or it may be in the formof a vector.

The vector may be a viral vector, preferably a retroviral, adenoviral,adeno-associated viral (AAV), herpes viral, alphavirus vector or anyother suitable vector as will be apparent to the skilled reader.Alternatively, the nucleic acid molecule may be in the form of anon-viral vector, preferably a plasmid vector. Many such vectors areknown and documented in the art (see, for example, Fernandez J. M. &Hoeffler J. P. in Gene expression systems. Using nature for the art ofexpression ed. Academic Press, San Diego, London, Boston, New York,Sydney, Tokyo, Toronto, 1998). Such vectors may additionally incorporateregulatory sequences such as enhancers, promoters, ribosome bindingsites and termination signals in the 5′ and 3′ untranslated regions ofgenes, that are required to ensure that the coding sequence is properlytranscribed and translated.

Alternatively, the nucleic acid molecule may be in the form polycationiccondensed DNA linked or unlinked to killed adenovirus alone, for examplesee Curiel (1992) Hum Gene Ther 3:147-154, or ligand linked DNA, forexample see Wu (1989) J Biol Chem 264:16985-16987. The nucleic acidmolecule may also be in the form of DNA coated latex beads.Alternatively, the nucleic acid molecule may be encapsulated inliposomes as described, for example, in WO95/13796, WO94/23697,WO91/14445 and EP-524,968. SA 91(24):11581-11585.

Antigen may be introduced into the animal by any suitable means.Preferably, the method of introduction involves injection. The animalsmay be immunised with the purified antigen or antigens intrasplenically,intravenously, intraperitoneally, intradermally or subcutaneously or byany other suitable means. The animals may be immunised with the purifiedantigen or antigens via more than one of these routes. For example, someof the purified antigen or antigens may be injected intraperitoneallyand the rest subcutaneously. The means of injection will depend on theantigen or antigens being injected. For example, in the case ofinjection with a nucleic acid molecule a hand-held gene transferparticle gun, as described in U.S. Pat. No. 5,149,655 can be used toinject the nucleic acid molecule.

In the case of a proteinaceous antigen, the dose of each antigen shouldpreferably be in the range of between 0.01 and 1000 micrograms.

Preferably, the method of the invention comprises the additional step ofsupplying the animal with a booster dose of some or all of the antigenswhich are introduced into the animal prior to the recovery of theantibody-producing cells. The animals may be given a booster 1-365 daysafter the first injection. Preferably, the animals are boosted 1 to 20times.

Preferably, the immunisation protocols used in the methodology of thepresent invention are short where more than one antigen is used in orderto prevent one antigen becoming immunodominant. Preferably, where theanimal is immunised with more than one antigen, it is injected with abooster of each antigen or combined booster of more than one antigen 3days after the first injection and a further booster 5 days after theinitial injection with spleen tissue or lymph nodes being removedbetween day 6 and day 15. Where a longer immunisation protocol isdesired, the animal may be injected, for example, with a booster of eachantigen or combined booster of more than one antigen 21 days after thefirst injection, with the spleen tissue or lymph nodes being removed onday 26.

Immunisation of the animal may be carried out with or withoutpharmaceutical carriers. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,lipid aggregates (such as oil droplets or liposomes), and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Additionally, these carriers may function as immunostimulatingagents (“adjuvants”). Immunisation of the animal may be carried out withor without adjuvants in addition to the pharmaceutical carriers.

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: (1) aluminium salts (alum), such as aluminiumhydroxide, aluminium phosphate, aluminium sulfate, etc; (2) oil-in-wateremulsion formulations (with or without other specific immunostimulatingagents such as muramyl peptides (see below) or bacterial cell wallcomponents), such as for example (a) MF59™ (WO90/14837; Chapter 10 inVaccine design: the subunit and adjuvant approach, eds. Powell & Newman,Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span85 (optionally containing TP-PE) formulated into submicron particlesusing a microfluidizer, (b) SAF, containing 10% Squalene, 0.4% Tween 80,5% pluronic-blocked polymer L121, and thr-MDP either microfluidized intoa submicron emulsion or vortexed to generate a larger particle sizeemulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components from the group consisting ofmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, suchas QS21 or Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may beused or particles generated therefrom such as ISCOMs (immunostimulatingcomplexes), which ISCOMS may be devoid of additional detergent e.g.WO00/07621; (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA); (5) cytokines, such as interleukins (e.g. IL-1, IL-2,IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.), interferons (e.g.gamma interferon), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (6) monophosphoryl lipid A (MPL) or3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454, optionally inthe substantial absence of alum when used with pneumococcal saccharidese.g. WO00/56358; (7) combinations of 3dMPL with, for example, QS21and/or oil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898,EP-A-0761231; (8) oligonucleotides comprising CpG motifs (Roman et al.,Nat. Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94,10833-10837; Davis et al., J. Immunol., 1998, 160, 870-876; Chu et al.,J. Exp. Med., 1997, 186, 1623-1631; Lipford et al., Eur. J. Immunol.,1997, 27, 2340-2344; Moldoveanu et al., Vaccine, 1988, 16, 1216-1224,Krieg et al., Nature, 1995, 374, 546-549; Klinmnan et al., PNAS USA,1996, 93, 2879-2883; Ballas et al., J. Immunol., 1996, 157, 1840-1845;Cowdery et al., J. Immunol., 1996, 156, 4570-4575; Halpern et al., Cell.Immunol., 1996, 167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988,79, 866-873; Stacey et al., J. Immunol., 1996, 157, 2116-2122; Messinaet al., J. Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996,157, 4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al.,J. Immunol., 1998, 160, 4755-4761; and Yi et al., J. Immunol., 1998,160, 5898-5906; International patent applications WO96/02555,WO98/16247, WO98/18810, WO98/40100, WO98/55495, WO98/37919 andWO98/52581) i.e. containing at least one CG dinucleotide, with5-methylcytosine optionally being used in place of cytosine; (9) apolyoxyethylene ether or a polyoxyethylene ester e.g. WO99/52549; (10) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol (WO01/21207) or a polyoxyethylene alkyl ether or estersurfactant in combination with at least one additional non-ionicsurfactant such as an octoxynol (WO01/21152); (11) a saponin and animmunostimulatory oligonucleotide (e.g. a CpG oligonucleotide)(WO00/62800); (12) an immunostimulant and a particle of metal salt e.g.WO00/23105; (13) a saponin and an oil-in-water emulsion e.g. WO99/11241;(14) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) e.g.WO98/57659; (15) other substances that act as immunostimulating agentsto enhance the effectiveness of the composition. Additional examples ofadjuvants that may be used include Montanide ISA 50, Hunter's TiterMax,and Gerbu Adjuvants.

Preferred antibody-producing cells for use in the invention include Bcells, T cells and stem cells. These antibody-producing cells for use inthe invention may be recovered by removal of any suitable cellularcomponents of the immune system from the animal. Preferably,antibody-producing cells are recovered from the animal by removal of thespleen, lymph nodes or bone marrow or portions thereof. These may berendered into a single cell suspension according to step b) of themethod of the invention via any suitable means. Preferably, spleentissue, lymph nodes or bone marrow removed from the animal are renderedinto a single cell suspension by mechanical disruption or enzymaticdigestion with proteases. Red cells may be removed from the cellsuspension by hypotonic lysis.

Preferably, the immortalized cell line specified in step c) of themethod of the invention is a hybridoma cell line produced by somaticfusion of the cells in the single cell suspension to myeloma cells.Cells in the single cell suspension are fused to myeloma cells with afusogen. Examples of myeloma cells which may be used include SP2, NS1and NS0. Preferably, the fusogen is PEG, a virus or a method ofelectrofusion (Zimmermann et al. 1990).

The hybridoma cells produced by the fusion of the single cell suspensionwith the myeloma cells should be cultured. Preferably, the hybridomacells are initially cultured in a selective media, such as Azaserinehypoxanthine or Hypoxanthine aminopterin thymidine, and are thentransferred to a non-selective media. Preferably, the hybridoma cellsare cultured on selective media for 7 days and are then transferred to anon-selective media for 3 days. This ensures that the growth rate of thecells increases prior to the screening step. Preferably, the stepsinvolved in hybridoma production are conducted robotically in order tospeed up the process. The Examples set out one way of conducting thesteps involved in hybridoma production robotically.

Alternatively, the immortalized cell line may be a cell line generatedby infection of cells in the single cell suspension with animmortalizing virus. Preferably, the immortalizing virus is Epstein-Barvirus (see, for example, Epstein Barr Virus Protocols, Eds. Wilson andMay, Humana Press; ISBN: 0896036901).

Step d) of the method of the invention comprises screening thesupernatant of the immortalized cell line, preferably a hybridoma cellline, against a protein chip comprising a candidate antigen with whichthe animal was immunised. As used herein, the term “protein chip” isused to encompass any microarray made up of a supporting means to whicha candidate antigen has been anchored.

Where just one purified antigen has been introduced into the animal,that purified antigen and no additional antigens may be anchored on theprotein chip. Where more than one purified antigen has been introducedinto the animal, each purified antigen may be displayed at a differentposition on the protein chip, preferably at a predetermined position.Each position on the protein chip may thus display a different antigen.Alternatively, the same antigen may be anchored to each position in arow or column of a protein chip with a different antigen being displayedin each row or column. In some cases, it may be desirable, even when theanimal has been immunised with more than one purified antigen, to have adifferent chip for each antigen. A protein chip may have a large number,such as between 1 and 1000 purified antigens, anchored at predeterminedpositions on a chip.

Any type of protein chip known in the art may be used in the method ofinvention. For example, the protein chip may be a glass slide to whichthe purified antigen or antigens are anchored. Where only one antigen isbeing tested, such a slide may be prepared simply by coating glassmicroscope slides with aminosilane (Ansorge, Faulstich), adding anantigen-containing solution to the slide and drying. Slides coated withaminosilane may be obtained from Telechem and Pierce for coating withthe purified antigen. Preferably, such a glass slide may be coated with(6-aminohexil) aminosilane.

Other types of protein chips which may be used in the method of theinvention include a 3D gel pad (Arkenov et al, 2000) and microwellchips. As will be apparent to the skilled reader, types of protein chipsthat have not yet been conceived but which are devised in the future maywell prove to be suitable for use in accordance with the presentinvention.

The term “protein chip” also includes microarrays of cells expressingdefined cDNAs (Ziauddin et al, 2001) referred to herein as “cell chips”.In this technique, mammalian cells are cultured on a glass slide printedin defined location with different cDNAs. Cells growing on the printedlocations take up and express the cDNAs. Cell chips are particularlyuseful when the animal has been injected with a cDNA or a cDNA libraryor with a recombinant virion or virions produced from a cDNA library, asdescribed above. In such cases, the proteins encoded by the cDNAsequences may not have been isolated. By injecting the animal with cDNAsencoding the proteins or recombinant virions expressing the cDNAs, it ispossible to produce monoclonal antibodies against the proteins expressedby the cDNAs. If the same cDNAs are expressed using a cell chip, theseantibodies will bind and the binding may be detected as described below.Providing that a nucleic acid sequence encoding the protein isavailable, the invention in this manner enables the detection andisolation of a monoclonal antibody against that protein which may beused to purify the protein itself.

For selection of antibodies, or selection of immortalised cell linesproducing such antibodies, the supernatant from the immortalised cellline or cell lines is spotted onto the protein chip or protein chips atdefined positions on the chip. Spotting of supernatants is preferablydone robotically, for example with a Genemachines Ominigrid arrayerusing Telechem pins. Preferably, the supernatants spotted onto theprotein chip or protein chips contain glycerol to minimise drying andfixing of the antibodies on the slide. For example, 0 to 99.9% glycerolmay be used. The chip is then washed to remove any unbound supernatant.At this stage, any monoclonal antibody produced by the immortalized cellline and hence in the supernatant may be bound to an antigen on thechip.

By using different elution conditions, the method allows the approximatequantification of the binding affinity of the monoclonal antibody forits binding partner. Elution agents that may be used include chaotropicagents such as guanidine hydrochloride or urea at concentrations between10 μmolar and 8 molar or ethylene glycol in an aqueous solution of 0.01%to 100% w/v. Elutions may also be carried out using aqueous ornon-aqueous solutions of glycine at concentrations of between 0.01 molarand a saturated solution (preferably 200 mM), at a pH of between pH9 andpH1, preferably pH3.2. High pH elutions may be carried out using aqueousor non aqueous solutions of triethylamine between 1 μmolar and asaturated solution, preferably 100 mM, at a pH of between pH8 and pHI3,preferably pH 11.5.

Step e) of the method of the invention involves selection of amonoclonal antibody that binds to the antigen. Preferably, this stepincorporates a detection step, such as by adding a marker which willbind to bound monoclonal antibody and indicate its presence.

Preferably, the marker is labelled with a label such as an enzymatic orfluorescent label that enables its presence to be detected. For example,the marker may be labelled protein A or labelled protein G. Protein A orprotein G may be labelled with a fluorescent label such as Cy3 or Cy5.Alternatively, protein A or protein G may be labelled with an enzymaticlabel such as biotin, the presence of which can be detected by thebinding of labelled strepavidin or avidin.

Preferably, the marker is an antibody that will bind to the firstantibody. Preferably this antibody is labelled with a label such as anenzymatic or fluorescent labels. Preferably this antibody is labeledwith fluorescent labels as this enables equipment developed for scanningof DNA chips to be used for detection.

Preferably, the step of detecting a monoclonal antibody bound to theantigen further comprises isotyping the monoclonal antibodies.Preferably, this step of detecting and isotyping the monoclonalantibodies comprises adding isotype-specific anti-immunoglobulinantibodies to said protein chip, wherein each anti-immunoglobulinantibody having a different isotype specificity has a different label,and detecting the presence of said labels. This method enables thesimultaneous detection of the monoclonal antibody and determination ofits isotype.

It will be appreciated that the method may employ as many differentisotype-specific anti-immunoglobulin antibodies, each with a differentlabel, as there are antibody isotypes in the animal which has beenimmunised. For example, if a mammal, such as a mouse, has been injectedwith an antigen, the step of detecting and isotyping monoclonalantibodies bound to the antigen may comprise adding an anti-IgA antibodylabelled with a first label, and/or an anti-IgD antibody labelled with asecond label, and/or an anti-IgE antibody labelled with a third label,and/or an anti-IgG1 antibody labelled with a fourth label, and/or ananti-IgG2a antibody labelled with a fifth label, and/or an anti-IgG2bantibody labelled with a sixth label, and/or an anti-IgG3 antibodylabelled with a seventh label, and/or an anti-IgG4 antibody labelledwith a eighth label, and/or an anti-IgM antibody labelled with a ninthlabel. Alternatively, the step of detecting and isotyping monoclonalantibodies bound to the antigen may comprise adding isotype-specificanti-immunoglobulin antibodies that bind to a subset of the possibleisotypes. Preferably, the isotype-specific anti-immunoglobulinantibodies comprise an anti-IgM antibody labelled with a first label andan anti-IgG antibody labeled with a second label. Preferably, the labelsare fluorescent labels.

Detection of the label indicates the presence of a monoclonal antibodybound to an antigen and is preferably done robotically. Where the labelis a fluorescent label, detection of the label and hence the presence ofthe monoclonal antibody may be done using equipment available forscanning protein chips. For example, scanning of the chips may be donewith a GenePix 4000B scanner (Axon Instruments, Inc.) or with a TecanLS200 or LS400 scanner. Scanning may be carried out with between 1 and 4lasers and combinations of filters to enable visualisation of multiplefluorescent labels. Preferably, visualisation of multiple fluorescentlabels is carried out simultaneously although it may be carried outsequentially.

Images may be obtained and analysed using appropriate software such asthe GenePix Pro software (Axon Instruments, Inc.), Chipskipper software(Schwager, Ansorge) or Tecan LS200 or LS400 software.

In order to ensure that the detection of a monoclonal antibody isreliable, the screening method preferably employs various controls. Forexample, in the case of a protein chip coated with one antigen, not onlywill the supernatants from the immortalized cell lines produced by themethod of the invention be spotted onto the protein chip but so willpositive and negative controls.

Positive controls may be in the form of previously tested monoclonalantibodies or commercially available polyclonals. Alternatively,positive controls may consist of diluted or undiluted serum previouslycollected from the immunized mouse either a suitable period after theboost or at the moment the animal is sacrificed for the collection ofthe source of B-cells.

Negative controls may be in the form of mock supernatants at definedpositions. Another level of control is determined by the fact that eachsupernatant is screened against several antigens. Signals obtainedagainst only one antigen are considered to be potential positivemonoclonal antibody containing supernatants.

Positive signals on the protein chip can be traced back to a particularimmortalised cell line enabling the monoclonal antibody to be isolatedaccording to step e) of the method of the invention. Furthercharacterisation of the antibodies identified can then be carried out.

Methods for carrying out further characterisation of the antibody mayinclude, for example, the further step of determining the specificity ofthe monoclonal antibodies identified. For example, a monoclonal antibodyidentified by the method of the invention may be used to scan a secondprotein chip having a large number of different proteins anchored to itssurface to establish if the monoclonal antibody binds specifically toone antigen. The scanning methods described above in the initialidentification of the protein may be used to scan for its specificity.The binding specificity and affinity of the monoclonal antibodiesproduced by the method of the invention may be further characterised byaltering the concentrations of antigen on protein chips or altering thestringency of eluting conditions, as described above.

According to a further embodiment of the first aspect of the invention,there is provided a method for producing an immortalised cell line thatproduces a monoclonal antibody of interest, said method comprising thesteps of:

-   -   a) introducing at least one candidate antigen into an animal;    -   b) recovering antibody-producing cells from said animal and        rendering these cells into a single cell suspension;    -   c) generating an immortalized cell line from said single cell        suspension;    -   d) screening the supernatant of said immortalized cell line        against a protein chip on which the candidate antigen is        displayed; and    -   e) selecting as said immortalised cell line, that which produces        a supernatant containing an antibody that binds to said        candidate antigen.

Immortalised cell lines produced by such a method are of immense utilityin the generation of antibodies with tailored specificities.

According to a particular embodiment of the first aspect of theinvention, there is provided a high-throughput method for producing aplurality of monoclonal antibodies, each of which binds to a differentcandidate antigen, comprising the steps of:

-   -   a) introducing a plurality of candidate antigens into an animal;    -   b) recovering antibody-producing cells from said animal and        rendering these cells into a single cell suspension;    -   c) generating immortalized cell lines from said single cell        suspension;    -   d) screening the supernatant of said immortalized cell lines        against one or more protein chips on which the candidate        antigens are displayed; and    -   e) selecting as said monoclonal antibodies, antibodies that bind        to said candidate antigens.

The candidate antigens are preferably purified candidate antigens, asdescribed above. Suitable procedures for introducing the candidateantigens into the animal, recovering antibody-producing cells,generating immortalized cell lines and screening the supernatants of theimmortalized cell lines are described above.

Prior art methods involve laborious and time-consuming procedures togenerate and screen for a monoclonal antibody against a single antigen.In contrast, this method enables the generation and high-throughputscreening of monoclonal antibodies against a plurality of antigenssimultaneously. The use of a protein chip to conduct high-throughputscreening of the antibodies is more efficient and more accurate than theuse of conventional assays and requires less candidate antigen.

Preferably, step e) of this embodiment further comprises isotyping themonoclonal antibodies, as described above. This provides an additionaladvantage over the prior art methods which do not disclose simultaneousdetection and isotyping of monoclonal antibodies.

According to a second aspect of the invention, there is provided amonoclonal antibody produced by a method of the invention. The inventionmay also be used to generate a bank of antibodies, for example, withspecificities encompassing an entire organismic proteome. Such a bank ofantibodies represents a further aspect of the invention.

According to a third aspect of the invention, there is provided animmortalized cell line, preferably a hybridoma cell line, which producesa monoclonal antibody according to the second aspect of the invention.This aspect of the invention also includes a bank of immortalized celllines, preferably a bank of hybridoma cell lines. The invention may alsobe used to generate a bank of hybridoma cell lines, for example, thatproduce antibodies encompassing an entire organismic proteome.

According to a fourth aspect of the invention, there is provided amethod for producing a plurality of monoclonal antibodies, each of whichbinds to a different purified candidate antigen, comprising introducinga plurality of purified candidate antigens into an animal.

Preferably, each candidate antigen is derived from a different source.By this is meant that each antigen is derived from a different protein,a different nucleic acid and so on. It is intended that methods ofantibody production that involve injecting an animal with differentfragments of the same protein are excluded from the scope of this aspectof the invention. For example, the purified candidate antigens may allbe proteinaceous substances provided that they are not all fragments ofthe same protein.

This method has an advantage over methods disclosed in the prior art inthat it enables the simultaneous production of more than one monoclonalantibody, each of which binds to a different purified candidate antigen.

The animal may be injected with the purified candidate antigens usingany of the techniques described herein. For example, the method of thisaspect of the invention may further comprise the steps of recoveringantibody-producing cells such as B cells, T cells and stem cells from animmunised animal, such as by removing spleen tissue, lymph nodes or bonemarrow, and rendering them into a single cell suspension. The method mayfurther comprise generating immortalized cell lines, preferablyhybridoma cell lines, from the cells the single cell suspension.Preferably, the method of this aspect of the invention comprises thesesteps and additionally comprises the steps of screening the supernatantsof the immortalized cell lines, preferably hybridoma cell lines, againsta protein chip or protein chips on which the candidate antigens aredisplayed; and selecting monoclonal antibodies that bind to the antigensand preferably isolating these and/or the immortalized cell lines thatproduce the monoclonal antibodies. Suitable procedures for generatingthe immortalized cell lines and subsequent screening of the supernatantsare the same as those described in above in connection with the methodof the first aspect of the invention. In particular, the step ofdetecting the monoclonal antibodies may involve simultaneous detectionof the monoclonal antibodies and determination of this isotype, asdescribed above. In addition, the method may comprise furthercharacterisation of the monoclonal antibodies, as described above.

The invention also provides a monoclonal antibody produced by a methodof this aspect of the invention. Again, this aspect of the invention maybe used to generate a bank of antibodies, for example, encompassingantibodies with specificity for protein in an entire organismicproteome. Such a bank of antibodies represents a further aspect of theinvention.

The invention also provides an immortalized cell line, preferably ahybridoma cell line, which produces a monoclonal antibody as describedabove. The invention may also be used to generate a bank of immortalizedcell lines, preferably a bank of hybridoma cell lines, for example, thatproduce antibodies encompassing an entire organismic proteome.

According to a fifth aspect of the invention, anti-idiotype antibodiesmay be generated that bind to a monoclonal antibody according to thesecond aspect of the invention. Anti-idiotype antibodies are useful asthey have a variable region that mimics the shape of the molecule towhich the original antibody was raised. Anti-idiotype antibodies maytherefore be useful in therapy as replacements for the molecules againstwhich the original antibody was raised. An anti-idiotype antibody may beproduced by employing the method of the first aspect of the invention orthe fourth aspect of the invention using a monoclonal antibody accordingto the second aspect of the invention as the purified candidate antigen.

Accordingly, this aspect of the invention provides a method of producingan anti-idiotype antibody that binds to a monoclonal antibody accordingto the second aspect of the invention, the method comprising using amonoclonal antibody according to the second aspect of the invention as apurified candidate antigen in a method of the first aspect of theinvention or the fourth aspect of the invention. The invention alsoincludes anti-idiotype antibodies generated by such methods.

According to a sixth aspect of the invention, anti-anti-idiotypeantibodies may be generated that bind to an anti-idiotype antibodyproduced according to the fifth aspect of the invention. Suchanti-anti-idiotype antibodies may be produced by employing the method ofthe first aspect of the invention or the fourth aspect of the inventionusing an anti-idiotype antibody as described above as the purifiedcandidate antigen This aspect of the invention thus provides a method ofproducing an anti-anti-idiotype antibody that binds to an anti-idiotypeantibody generated according to the fifth aspect of the invention, themethod comprising using an anti-idiotype antibody as described above asa purified candidate antigen in a method of the first aspect of theinvention or the fourth aspect of the invention.

Various aspects and embodiments of the present invention will now bedescribed in more detail by way of example. It will be appreciated thatmodification of detail may be made without departing from the scope ofthe invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Whole image of scanned chip, where green and red spots representpositive IgG and IgM producing supernatants respectively. Close ups areto show details of specific areas of chip where good spots are to befound.

FIG. 2: Comparison between chip analysis and ELISA screen. First imageis negative sample (Ia<0.5), while others are positive. Average Ic:Average total intensity of spot on 3 chips. Ia: Average contribution ofspot to sum of intensities over three chips (%).

FIG. 3: Comparison of contribution to total intensity (%) of culturesupernatants screened by ELISA (▪) or by chip analysis (□) for bindingto B5 antigen (FIG. 3A). The background values are shown in FIG. 3B. Anumber of positive supernatants identified by chip analysis and/or ELISAare shown.

FIG. 4: Comparison of contribution to total intensity (%) of culturesupernatants screened by ELISA (▪) or by chip analysis (□) for bindingto B5 antigen (FIG. 4A). The background values are shown in FIG. 4B. Asingle positive supernatant identified by chip analysis and ELISA isshown.

FIG. 5: Comparison of contribution to total intensity (%) of culturesupernatants screened by ELISA (▪) or by chip analysis (□) for bindingto Ket94/95 antigen (FIG. 5A). The background values are shown in FIG.5B. A number of positive supernatants identified by chip analysis and/orELISA are shown.

FIG. 6: Comparison of contribution to total intensity (%) of culturesupernatants screened by ELISA (▪) or by chip analysis (□) for bindingto Ket94/95 antigen (FIG. 6A). The background values are shown in FIG.6B. No positive supernatants were identified by either ELISA or chipanalysis.

EXAMPLES Example 1 Immunization with 10 Protein Antigens

Immunization

An 8-week old female Balb/c mouse was injected intrasplenically with 10μg of each of 10 protein antigens in 100 μl phosphate buffered saline(PBS). On the third day following (day 3) the mouse was injectedintraperitoneally with lug of each of the same 10 protein antigens in100 μl PBS. On day 5, the same mouse was injected intravenously with 0.1μg of each of the same 10 protein antigens. On Day 8 the mouse waskilled by cervical dislocation and the spleen removed and collected intoDulbecco's Modified Eagle's Medium (DMEM: Life Technologies Inc.).

Fusion

All steps are performed under sterile or aseptic conditions in a laminarflow hood. The spleen was rendered into a single-cell suspension bymechanical disruption between two frosted-end glass microscope slides.The suspension was filtered into a 50 ml bar-coded conical-bottomed tube(BD Falcon) through a 70 μm nylon cell strainer (BD Falcon) andtransferred to the robotic system.

Separately, SP2 myeloma fusion partners (ATCC) were cultured for fivedays prior to fusion in HM20 (DMEM, 20% Defined fetal bovine serum(Hyclone Defined), 10 mM L-Glutamine, 50 μM Gentamicin) and on the dayof the fusion were transferred to HM20/HCF/2×OPI (HM20 containing 10%Hybridoma Cloning Factor (Origen) and 2% OPI cloning supplement (Sigma))for at least one hour at 37° C. in a 5% CO₂ incubator.

The bar codes were read by a bar code reader and the 50 ml Falcon tubeloaded into the rotor by the RoMa arm on the genesis Freedom system(Tecan). The rotor was loaded into the centrifuge through the workdeck.

The tube was centrifuged at 10 g for 10 mins at room temperature (RT)and the rotor extracted from the centrifuge. The tube was extracted fromthe rotor and the bar code was again read to distinguish from thebalance tube. Cells were resuspended in 5 ml Red Cell Lysis Buffer(Sigma) for 9 minutes at RT. HM20 was added to 50 ml and the tube onceagain centrifuged for 10 min at RT with no brake. The supernatantsolution was aspirated to waste and the cells resuspended in DMEMpreheated to 37° C. Cells were washed twice more by steps ofcentrifugation and resuspension. 50 μl of cell suspension wererobotically pipetted to a 1.5 ml microcentrifuge tube. Cells werecounted using a haemocytometer counting chamber.

Simultaneously the SP2 cells were washed three times in a similarfashion and a similar aliquot (50%1) “handed off” to a 1.5 ml tube forhaemocytometric counting. SP2 myelomas and spleen cells were mixed at aratio of 1:5 (SP2:Spleen) and again centrifuged at 100 g for 10 min withno brake.

The supernatant solution was entirely aspirated to waste andPolethyleneglycol 1500 in 50% HEPES (PEG: Roche Molecular Biochemicals)pre-heated to 37° C. was robotically pipetted smoothly and progressivelyover 1 min with rotation at 450 rpm on a Te-shake shaker (Tecan AG) toensure even mixing. The cell/PEG mixture was incubated for 1 min at 37°C. with gentle agitation. 1 ml of DMEM was similarly added over 1 min at37° C. with similar agitation. The mixture was incubated for 1 min at37° C. with gentle agitation. A further 1 ml of DMEM was roboticallyadded over 1 min at 37° C. with gentle agitation and incubated similarlyfor a further minute. 7 mls of HM20 were robotically added over 3 min at37° C. with gentle agitation. The Tube was then spun at 90 g for 5 minwith brake. The supernate was aspirated to waste and the pelletresuspended in 20 ml of HM20/HCF/OPI/AH (HM20/HCF/OPI plus 10% AzaserineHypoxanthine (Sigma).

The conical tube was again placed on the robot workdeck and thepost-fusion cell slurry was aspirated by each of the 8 wide-bore pipettetips of the liquid handling arm of the robot. 200 μl of the cell slurrywas then pipetted into each well of a 96-well deep well plate (GreinerMasterblock).

The deep-well plate was then robotically transferred to a TeMo 96-wellpipetting robot integrated onto the Genesis work-deck and used as asource plate to plate out into the 20 sterile 96-well tissue cultureplates.

The post-fusion mixture was then robotically plated out into 20 96-wellsterile plates (Nunc) sourced from a carousel attached and integrated tothe robot at 100 μl/well and robotically transferred to an integrated37° C. incubator with 10% CO₂ through the integrated airlock. Plateswere stored in a carousel contained with the incubator.

Cell Culture

On the third day following the fusion cells were robotically transportedfrom the incubator to the work deck and a further 100 μl HM20/HCF/OPI/AHwas robotically added. The plates were then robotically returned to theincubator.

On day 7 the plates were once again similarly transported from incubatorto work deck and 200 μl/well of culture supernates was aspirated towaste and replaced with 150 μl fresh HM20/HCF.

On day 11 the plates were again robotically transported to the work deckand 30 μl of supernate was collected from each well (Temo head: TecanInc.) and transferred to 384-well plates supplied to the workdeck by acarousel plate stacker (Tecan Inc).

Microarray Screening

Aminosilane coated glass slides were homogeneously coated with purifiedantigen by dropping 40 μl of ddH₂O containing 1-5 μg of antigen andcovering with a 22*60 mm coverslip for 60 min in a humid chamber at RT.

Coated slides were rinsed briefly in PBS and blocked for 60′ in 5% milkin PBS, 0.1% Tween, then washed for 10′ in PBS. The chips were thendried by centrifugation, 10″ at 2000 rpm.

Culture supernatants were consolidated into 384 well plates using aBeckman Biomek FX robot.

Culture supernatants were printed singularly onto three identicalantigen-coated slides at a density of 9600 spots per chip and a spotsize of ˜120 μm using a GeneMachines OmniGrid microarray printer.

The microarray chips were incubated in a humid chamber for 60′, at RTand then washed 5×5′ in PBS-0.1% Tween (PBST) 40 μl of Cy3 conjugatedgoat anti-mouse IgG-specific and Cy5 conjugated goat anti-mouseIgM-specific antibodies were diluted 1:1000 in PBST, mixed and applieduniformly to the chips and covered with 22×60 mm coverslips andincubated in a humid chamber, for 30′ at RT. The chips were then washed2×10′ in PBST, 2×10′ in PBS and 1×10′ in ddH₂O. The chips were dried bycentrifugation at 2000 rpm for 10″.

Hit-Picking

Chips were scanned with a GenePix 4000B scanner (Axon Instruments), at aresolution of 10 um.pixel⁻¹. PMT voltages were 540V and 610V, for theCy3 and Cy5 channels respectively. Both lasers were set at 100%intensity. Each scanned chip was assigned a fitted grid, and all spotswere analysed by the GenePix Pro 3.0 (Axon Instruments).

All chips were analysed by the GenePix Pro 3.0 software and wecollected, for each chip, the information relative to the correctedintensity (I_(c)) of each spot (Median of intensities—Background), forthe Cy3 and Cy5 channels. From this data, we obtained, for each chip,the contribution of each spot to the total Corrected Intensity of eachchannel (

I_(c)/Σ_(t) ⁹⁶⁰⁰I_(c)

×100). The three values of each channel were averaged for each spot, andthis Average Corrected Intensity (I_(a)) was used for the final analysisof the dataset. We considered as “likely to be positive” samples theones that have a value above 0.5% and as “sure positives”, all thesamples showing a value superior to 1.5%.

Post Screening Processing

Cells from positive wells were resuspended in the well and 20 μltransferred to a 96-deep-well plate previously filled with 1.5 mls ofHM20/HCF and returned to the incubator for 48 hrs at 37° C., 5% CO₂.

1.4 ml of culture each supernate was transferred to another deep wellplate and the remaining 100 μl used to resuspend the cells which werethen transferred to a freezing vial containing 90% Foetal bovineserum/10% DMSO (Sigma) and transferred to −80° C. for 2 hrs and fromthere to liquid nitrogen store. Culture supernate was then used forevaluation and further characterization of the generated monoclonalantibody.

Results:

The results of microarray screening are shown in FIG. 1. This Figuredemonstrates that a number of positive monoclonal antibodies weredetected as binding to candidate antigens on the slide. The green spotsare IgG monoclonal antibodies which bind to the candidate antigens whilethe red spots are IgM monoclonal antibodies which bind to the candidateantigens. FIG. 1 thus demonstrates that the method of the invention canbe used to simultaneously identify monoclonal antibodies that bind tothe candidate antigens and the isotypes of those monoclonal antibodies.

When a comparative experiment was conducted using ELISA in the place ofa protein chip, a number of monoclonal antibodies identified as bindingto candidate antigens in the microarray screening were not identified asbinding to candidate antigens using ELISA, as shown in FIG. 2 (seecomparison of microarray results with ELISA results). A supernatantwhich was found to be negative using microarray screening (Ia:0.234) wasalso found to be negative using ELISA. However, of four supernatantsfound to be positive using microarray screening (Ia: 5.18, 1.96, 3.64and 2.02), only two were found to be positive using ELISA (see FIG. 2).It is important to note how the supernatants that were negative in theELISA experiment can be actual positive samples as detected by the moreaccurate and sensitive microarray approach.

Example 2 Immunization with Nine Antigens

Immunization:

A mouse was injected with 25 μg of nine antigens, including 25 μg of afusion of the antigens B5 and Ket94/95, each antigen being mixed with 10μg CpG DNA and adsorbed onto alum adjuvant (Imject Alum from Pierce).Half of each antigen was administered intraperitoneally and halfsubcutaneously.

The mouse was boosted 21 days later with 10)1 g of each antigen mixedwith 10)1 g of CpG DNA and adsorbed onto alum adjuvant, half of whichwas administered intraperitoneally and half subcutaneously.

Five days after the boost, the spleen was removed.

Fusion and Cell Culture

The fusions and cell culture were performed as described in Example 1.

Microarray Screening of Antibodies Against B5 and Ket94/95.

An aminosilane glass slides was homogenously coated with purified B5 bydropping 40 μl of ddH₂O containing 5 μg of purified B5 and covering witha 22*60 mm coverslip for 60 min at room temperature. The same procedurewas used to produce an aminosilane glass slide homogenously coated withpurified Ket94/95.

The coated slides were rinsed, blocked, washed and dried, as describedin Example 1, except that 3% BSA in PBS was used in place of 5% milk inPBS to block the slide.

Culture supernatants were consolidated into 384 well plates, asdescribed in Example 1 and were printed in triplicate onto the slidecoated with B5 and the slide coated with Ket94/95 at a density of around16000 spots per chip and a spot size of ˜150μ m using a Microgrid II 610microarray printer (ApogentDiscoveries).

The microarray chips were incubated and Cy3 conjugated goat anti-mouseIgG-specific and Cy5 conjugated goat anti-mouse IgM-specific antibodieswere applied to the chips as described in Example 1.

Hit Picking

The chips were scanned using the same procedure as in Example 1. AnAverage Corrected Intensity (Ia) was calculated for each set oftriplicate culture supernatants.

Comparison of Microarray Screening and ELISA

A comparative experiment was conducted in which each culture supernatantwas checked by ELISA. Each culture supernatant was added to a wellcontaining 200 ng of B5 or Ket94/95 antigen and the presence of amonoclonal antibody that bound to the antigen in the culture supernatantwas detected using a conventional ELISA.

One week was required to screen 5376 culture supernatants using ELISA,producing 5376 results, one for each supernatant. In contrast, it tookunder 48 hours to screen the same 5376 culture supernatants intriplicate using a microarray chip, producing 16128 results,demonstrating the efficiency of the microarray screening compared toELISA.

Furthermore, only 5 μg of B5 antigen and 5 μg of Ket94/95 was requiredto carry out the microarray screening, 5 μg antigen per chip. Incontrast, 96%1 g of antigen was required for each ELISA plate and fiveELISA plates were required to screen each antigen. Screening by ELISAwas thus significantly more costly and time-consuming that microarrayscreening.

The data obtained for each ELISA plate was normalised to provide apercentage contribution to total intensity for each culture supernatant.The averaged replicate intensities for the same culture supernatantsobtained by microarray screening were also normalised to allowcomparison with the ELISA data.

When culture supernatants were screened using ELISA for monoclonalantibodies binding B5, 53 positive supernatants were identified overfive ELISA plates. Screening of the same culture supernatants usingmicroarray screening identified 48 positive supernatants, 90.57% of thesupernatants identified by ELISA. Microarray screening also identified 4novel positives that were not identified by ELISA.

When culture supernatants were screened using ELISA for monoclonalantibodies binding Ket94/95, 15 positive supernatants were identified ona single ELISA plate. Screening of the same culture supernatants usingmicroarray screening identified 13 positive supernatants, 88.66% of thepositive supernatants identified by ELISA. Microarray screening alsoidentified 8 novel positives that were not identified by ELISA.

It is clear from these results that microarray screening is at least aseffective as ELISA at identifying monoclonal antibodies that bind aspecific antigen. Indeed the identification of positive supernatants notidentified by ELISA suggests that microarray screening is more sensitivethan ELISA. Microarray screening further had the significant advantagethat it allowed simultaneous determination of the IG or IgM isotype ofthe monoclonal antibodies identified.

FIG. 3A shows the normalised values of percentage contribution to totalintensity for each culture supernatant in an ELISA plate (▪) containingpositive samples that bind B5 compared to the normalised values ofpercentage contribution for the same culture supernatants obtained bymicroarray screening of a B5-coated slide (O). FIG. 3B shows the levelof background noise in these experiments. It can be seen that positivesupernatants showed a greater percentage contribution to total intensityusing microarray screening compared to ELISA. As a result, there was agreater difference between background noise and a positive supernatantin microarray screening compared to ELISA, enabling positivesupernatants to be identified more easily and more accurately.

FIG. 4A compares the normalised values of percentage contribution tototal intensity for each culture supernatant in an ELISA plate (▪)containing a single positive sample that binds B5 compared to thenormalised values of percentage contribution for the same culturesupernatants obtained by microarray screening on a B5-coated slide (O).The level of background noise is shown in FIG. 4B and it can be seenthat positive sample was more readily detectable above the backgroundnoise using microarray screening compared to ELISA.

FIG. 5A compares the normalised values of percentage contribution tototal intensity for each culture supernatant in the ELISA plate found tocontain positive supernatants that bind KET94/95 (▪) compared to thenormalised values of percentage contribution for the same culturesupernatants obtained by microarray screening on a KET94/95-coated slide(□). The positive supernatants were more readily detectable above thebackground noise using microarray screening compared to ELISA, as shownin FIG. 5B.

FIGS. 6A compares the data obtained from an ELISA plate (▪) in whichthere were no positive supernatants to data obtained using microarrayscreening (□) of the same culture supernatants. As shown in FIG. 6B, thereadings in both cases were due to background noise.

These results demonstrate that the method of the invention can be usedto simultaneously identify monoclonal antibodies against more than oneantigen. The use of microarray screening in the method of the inventionis quicker, cheaper and more accurate than the use of conventionalantibody screening methods, such as ELISA.

References

-   Kilpatrick et al (1997) Hybridoma 16: 381-389-   Kohler G, Milstein C (1975) Nature 7: 256:485-7-   Lindley et al (2000) J Immunol Methods 234: 123-135-   Ziaudin J, Sabatini D (2001) Nature 411:107-110-   Zimmermann U. (1990) J Immunol Methods Nov 6;134(1):43-50

1. A method for producing a monoclonal antibody, said method comprisingthe steps of: a) introducing at least one candidate antigen into ananimal; b) recovering antibody-producing cells from said animal andrendering these cells into a single cell suspension; c) generating animmortalized cell line from said single cell suspension; d) screeningthe supernatant of said immortalized cell line against a protein chip onwhich the candidate antigen is displayed; and (e) selecting as saidmonoclonal antibody, an antibody that binds to said candidate antigen.2. The method of claim 1 wherein said animal is a mouse, a rat, a guineapig or a rabbit.
 3. The method of claim 1 or claim 2 wherein saidcandidate antigen is a purified candidate antigen.
 4. The method ofclaim 3 wherein between one and fifty different purified candidateantigens are introduced into the animal.
 5. The method of claim 4wherein between 0.001 and 1000 micrograms of each antigen is introducedinto the animal.
 6. The method of claim 1 comprising the additional stepof supplying the animal with a booster dose of some or all of theantigens which were introduced into the animal prior to the removal ofantibody-producing cells.
 7. The method of claim 1 wherein theantibody-producing cells are B cells, T cell or stem cells.
 8. Themethod of claim 1 wherein the antibody-producing cells are recovered byremoval of spleen tissue, lymph nodes or bone marrow of the animal. 9.The method of claim 1 wherein the immortalized cell line is a hybridomacell line produced by somatic fusion of the cells in the single cellsuspension to myeloma cells.
 10. The method of claim 1 wherein saidprotein chip is a plain-glass slide, a 3D gel pad chip, a microwell chipor a cell chip.
 11. The method of claim 1 wherein the step of detectingthe monoclonal antibodies bound to the antigens further comprisesisotyping the monoclonal antibodies.
 12. The method of claim 11 whereinsaid step of detecting and isotyping the monoclonal antibodies comprisesadding isotype specific anti-immunoglobulin antibodies to said proteinchip, wherein each anti-immunoglobulin antibody having a differentisotype specificity has a different label, and detecting the presence ofsaid labels.
 13. The method of claim 1 further comprising assessing thespecificity with which each isolated monoclonal antibody binds to anantigen using a protein chip comprising said antigen.
 14. Ahigh-throughput method for producing a plurality of monoclonalantibodies, each of which binds to a different candidate antigen,comprising the steps of: a) introducing a plurality of candidateantigens into an animal; b) recovering antibody-producing cells fromsaid animal and rendering these cells into a single cell suspension; c)generating immortalized cell lines from said single cell suspension; d)screening the supernatant of said immortalized cell lines against one ormore protein chips on which the candidate antigens are displayed; and e)selecting as said monoclonal antibodies, antibodies that bind to saidcandidate antigens.
 15. A high-throughput method for producing aplurality of monoclonal antibodies, each of which binds to a differentcandidate antigen comprising the steps of: a) introducing a plurality ofcandidate antigens into an animal; b) recovering antibody-producingcells from said animal and rendering these cells into a single cellsuspension; c) generating immortalized cell lines from said single cellsuspension; d) screening the supernatant of said immortalized cell linesagainst one or more protein chips on which the candidate antigens aredisplayed; and e) selecting as said monoclonal antibodies, antibodiesthat bind to said candidate antigens, which further comprises any of thesteps recited in claim
 1. 16. A method for producing an immortalizedcell line that produces a monoclonal antibody of interest, said methodcomprising the steps of: a) introducing at least one candidate antigeninto an animal; b) recovering antibody-producing cells from said animaland rendering these cells into a single cell suspension; c) generatingan immortalized cell line from said single cell suspension; d) screeningthe supernatant of said immortalized cell line against a protein chip onwhich the candidate antigen is displayed; and e) selecting as saidimmortalized cell line, that which produces a supernatant containing anantibody that binds to said candidate antigen.
 17. An immortalized cellline isolated by the method of claim
 16. 18. A method for producing aplurality of monoclonal antibodies, each of which binds to a differentpurified candidate antigen, comprising introducing a plurality ofpurified candidate antigens into an animal, each purified candidateantigen being derived from a different source.
 19. A method forproducing a plurality of monoclonal antibodies, each of which binds to adifferent purified candidate antigen comprising introducing a pluralityof purified candidate antigens into an animal, each purified candidateantigen being derived from a different source, which further comprisesany of the steps recited claim
 1. 20. A monoclonal antibody isolated bythe method of claim
 1. 21. An antibody according to claim 20 which is ananti-idiotype antibody.
 22. An antibody according to claim 21 which isan anti-anti-idiotype antibody.
 23. An immortalized cell line producinga monoclonal antibody of claim
 20. 24. An immortalized cell lineaccording to claim 23 which is a hybridoma cell line.
 25. A bank ofantibodies according to claim
 20. 26. A bank of immortalized cell linesaccording to claim 15.